Polynucleotide separations have become increasingly important in recent years. Separating polynucleotide species contained in a sample is useful, for example, in the detection andlor quantification of DNA that is the product of amplification reactions, and in the detection of variant DNA (e.g. polymorphisms or mutations). Due in large part to the complex structures and large sizes of such molecules, however, none of the separation techniques devised to date have proven wholly satisfactory.
Traditionally, polynucleotide separations have been performed using electrophoretic methods, such as slab gel electrophoresis or, more recently, capillary electrophoresis. Generally, these methods involve passing an electric current through a medium into which a mixture containing the species of interest has been injected. Each kind of molecule travels through the medium at a different rate, depending upon its electrical charge and size. Unfortunately, the electrophoretic methods are associated with certain disadvantages. For example, slab gel electrophoresis suffers the drawbacks of relatively low speed, difficulty in detection of samples, poor quantitation, and is labor intensive. Although faster and less labor intensive, capillary electrophoresis has suffered from irreproducibility of separations due to changes in the capillary performance and relatively poor quantitation.
Liquid chromatography has provided an alternative to the electrophoretic separation methods. Generally, the liquid chromatographic methods rely on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the components in a mixture. A column tube, or other support, holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster. As the components elute from the column they can be quantified by a detector and/or collected for further analysis.
Typical stationary phases, and their interactions with the solutes, used in liquid chromatography are:
STATIONARY NAME PHASE INTERACTION Size- Porous inert Samples are separated by virtue of their size Exclusion particles in solution; different sized molecules will have different total transit times through the column. Ion- Ionic groups Sample ions will exchange with ions already Exchange on a resin on the ionogenic group of the packing; retention is based on the affinity of different ions for the site and on a number of other solution parameters (pH, ionic strength, counterion type, etc.). Reverse- Non-polar Samples are separated based on hydrophobic Phase groups on interactions with the stationary phase. a resin
Although the liquid chromatographic methods have some advantages over electrophoretic separation techniques, they are not without their shortcomings. Size-exclusion chromatography suffers from low resolution, as, typically, DNA molecules must differ in size by 50-100% in order to obtain acceptable resolution. Although ion-exchange chromatography offers higher resolution, it can be affected by anomalous elution orders based on DNA sequence composition. Also with ion-exchange chromatography, the eluted DNA is often heavily contaminated with nonvolatile buffers that can further complicate sample recovery. Reverse-phase chromatography is capable of relatively high resolution but, unless special small-diameter particulates are used as the stationary phase, it cannot be performed at a very high speed. Silica-based particulates used in reverse-phase chromatography have suffered from low speed and instability at high pH conditions. Polymeric particulates have not been able to provide a high recovery of sample components or high separation speeds.
With particular regard to DNA separations, a technique known as reverse-phase ion-pair high performance liquid chromatography (RP-IP HPLC) has provided a limited amount of relief from some of the problems discussed above. For example, RP-IP HPLC avoids the problem of anomalous elution orders often encountered with packed beds bearing strong-anion exchangers. In RP-IP HPLC, the stationary phase typically consists of discrete particles bearing hydrophobic surface groups that are packed into a column. The eluent contains a cationic species, such as triethylammonium ion (0.1M), capable of interacting with the negatively charged phosphate groups on DNA and also with the hydrophobic surface of the particles in the column. Thus, the cationic species can be thought of as a bridging molecule between DNA and the column. As the mobile phase is made progressively more organic, e.g., with increasing concentration of acetonitrile, the DNA fragments are eluted in order of size.
Despite the advantages of RP-IP HPLC for DNA separations, the technique nevertheless suffers from problems common to all liquid chromatographic techniques wherein small particles (e.g., beads) are packed to form a bed in a column tube. For example, the production of particulate separation media can be complex and time-consuming. Once prepared, it can be difficult to pack the particles in columns in a reproducible and efficient manner. In particular, it has been difficult to pack efficient columns of small dimensions, such as columns less than 1 mm in diameter. Columns having norcclrcular cross-sections, such as polygonal cross-sections (e.g., thin-layer or rectangular), would be extremely difficult to prepare from particulate packing materials. Also, columns based on packed beads can fail due to shifting of packing material and the development of channels or voids. As an additional disadvantage, the use of small particles often leads to high column operating pressures, which necessitate column tubes, pumps, injectors and other components capable of containing fluid pressures of 3,000 psi or greater.
Current chromatographic theory predicts that, for beds of packed particles, separation efficiency will be determined by the diffusional distance for mass transfer of sample molecule to the stationary phase. This effect is easily modeled based on the known geometry of the particles used in such beds. According to such theory, maximum resolution will occur when the stationary phase has pore diameters of at least 3 times the Stokes' diameter of the molecule to be separated. This theory is based on the assumption that the molecule will be transported to the stationary phase by a diffusive process and that smaller pores would cause hindered diffusion. For DNA separations, this means that for a sample of 1,000 base pairs in size, a pore diameter of at least 1 micrometer will be needed. The need for such large pores practically eliminates the use of porous particulate materials packed in a support for the separation of DNA, as such matrices lack the required physical strength to be used at the operating pressures encountered in high performance liquid chromatography.
Nonporous particulate materials are preferred for DNA separations due to the reduction in diffusion distance compared to porous packings. For nonporous packings, the pore diameter is determined by the interstitial dimensions of the packed bed. The interstitial dimension is approximately 1/3 of the diameter of a spherical packing particle. A lower limit to the pore diameter may be imposed by the need to avoid trapping or shearing of larger DNA molecules. A higher limit will be imposed by the loss of efficiency that occurs when using larger particles. Separation using larger particles would be advantageous because of the lower operating pressures involved, but the loss in separation efficiency is too great.
As a further disadvantage, spherical packings can be packed into stable beds only at densities approximating an interstitial void fraction of about 0.4. Although somewhat more variable, nonspherical packings are also packed optimally at void fractions of about 0.4. Moreover, packed-particulate beds have fixed surface areas, fixed interstitial distances, and fixed pressure drops determined by the particle diameter of the packing material. Since DNA has a physical size approximating 0.34 micron per 1,000 base pairs of length and efficiency will be highest when the interstitial distance is 3-10 times the Stokes' diameter of the molecules being separated, a column designed for DNA of 1,000 base pairs could require an interstitial distance of 1-3 microns. Columns with packing of 3-10 micrometers in diameter would provide such distances. A column packed with 3 micrometer packings will have high operating pressures in use, whereas a column packed with 10 micrometer packings will show limited efficiency due to longer diffusional paths and lower binding capacity due to a decreased surface area. While columns packed with very small particles, such as 1 micrometer diameter, could be useful for samples up to 300 base pairs in length, column operating pressures would be very high and DNA larger than 300 base pairs could be sheared or trapped. A typical column of packed beads uses particles of 2.1 micrometers in diameter and has high operating pressure under typical use.
The above theory relating to beds of packed particles is not particularly useful for predicting the behavior of macromolecules in continuous monolithic beds, where mass transport may be a combination of diffusive and convective processes.
The present invention, which teaches monolithic beds for resolving mixtures containing polynucleotides, is based in part on the discovery that monoliths provide reduced pressure drops corresponding to the use of largeparticle stationary phases while maintaining the separation resolution of columns packed with small spherical particles. More particularly, it has been discovered that reversed-phase monolithic matrices can provide an improved method for the high speed separation of DNA molecules and that such separations can be performed with high resolution at greatly reduced operating pressures compared to previously available methods. This surprising finding now permits the high-resolution separation of polynucleotides under conditions not possible with preexisting technology. Moreover, the monolithic columns of the present invention can be constructed with stationary phase geometries significantly different than those available with packed beds. The effects of such novel geometries on the separation of macromolecules have not been predicted so far by current chromatographic theory.
The monolithic columns of the present invention provide all of the advantages of the previous best technology for polynucleotide separations (i.e., packed beds of alkylated nonporous polymer beads), without the need to tediously prepare beads and pack them into efficient columns. The columns produced by the current invention are easily prepared using simple processes and once prepared, cannot fail through shifting within a packed bed because there are no individual beads to shift position.
In addition to the improved ease of manufacturing of the new columns and lack of bead shifting, the monolithic columns described herein provide a surprising advantage over the existing technology in that they can offer at least 58% better resolution than that expected for a column of packed spheres when normalized for operating pressures.